Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease or motor neuron disease (MND), is one of several neurodegenerative diseases of the central nervous system. ALS is the most common adult onset motor neuron disease, affecting one in every 20,000 individuals, with an average age of onset of 50-55 years. ALS is characterized by rapidly progressive degeneration of motor neurons in the brain, brainstem, and spinal cord (Cleveland, 2001). The median survival of patients from time of diagnosis is five years.
ALS exists in both sporadic and familial forms. Familial ALS (FALS) comprises only 5-10% of all ALS cases. Over the last decade, a number of basic and clinical research studies have focused on understanding the familial form of the disease, which has led to the identification of eight genetic mutations related to FALS. Transgenic mice expressing point mutants of the Cu/Zn superoxide dismutase-1 (SOD1) gene develop an age-dependent progressive motor weakness similar to human ALS due to a toxic gain of function (Rosen, 1993; Rosen, 1994; Borchelt, 1994).
These genetic mutations, however, do not explain sporadic ALS (SALS). The pathogenesis of SALS is multifactorial. A number of different model systems, including SOD1 transgenic mice, in vitro primary motor neuron cultures or spinal cord slice cultures, in vivo imaging studies, and postmortem examination of tissue samples, have been utilized to understand the pathogenesis of ALS (Subramaniam, 2002); Nagai, 2001; Menzies, 2002; Kim, 2003; Ranganathan, 2003). Although these studies have yielded therapeutic targets and several clinical trials, there are no drugs that delay disease onset or prolong long-term survival of ALS patients. Riluzole (Rilutek®, Aventis), a glutamate antagonist, currently is the only FDA-approved medication available to treat ALS. Riluzole, however, extends life expectancy by only a few months (Miller, 2003). Creatine and a-tocopherol have shown some efficacy in relieving the symptoms of ALS in SOD1 transgenic mice, but exhibit minimal efficacy in human ALS patients (Groeneveld, 2003; Desnuelle, 2001).
Studies have been performed which have identified early protein biomarkers for ALS, using mass spectrometry based proteomics of cerebrospinal fluid (CSF) and spinal cord samples of human subjects (see U.S. Patent Application 10/972,732, published as US 2005-0148026 A1, the disclosure of which is incorporated herein). For example, three neuroendocrine proteins (transthyretin, 7B2, and cystatin C) that exhibit alterations early in the disease pathogenesis in humans were identified in a proteomics analysis.
Transthyretin regulates thyroid function and retinoic acid signaling in the brain by binding T4 and retinol-binding protein, respectively, and binds other proteins including the amyloid beta peptide (Aβ) to help sequester and prevent amyloid deposition (Palha, 2002; Bernstein, 2002; Tsuzuki, 2000). Retinoic acid signaling is an important component of neural plasticity and regeneration, and absence of retinoids can induce motor neuron disease in rats and decreased retinoid signaling has been observed in sporadic ALS patients (Mey, 2004; Corcoran, 2002).
Transthyretin (TTR) is a protein made by motor neurons in the spinal cord and choroid plexus cells that line the ventricles of the nervous system and produce the CSF. Transthyretin regulates thyroid function and retinoic acid signaling in the brain by binding T4 and retinol-binding protein, respectively, and binds other proteins including the amyloid beta peptide (Aβ) to help sequester and prevent amyloid deposition (Palha, 2002; Bernstein, 2002; Tsuzuki, 2000). T4 is a critical component of thyroid function and can also function to impede cell cycle progression and induce cell differentiation. Direct administration of T4 into a transgenic model for multiple sclerosis enhances remyelination and is neuroprotective for axonal pathology (Fernandez et al, 2004). Retinoic acid is a known antioxidant, and oxidative injury is one proposed mechanism for motor neuron cell death in ALS. Decreased levels of TTR also have been associated with increased brain levels of metals such as lead in the nervous system and metal toxicity induced neurodegeneration (Zheng et al., 2001). TTR also has been recently reported to have direct neuroprotective functions in neurons. The addition of TTR to neurons was found to be protective to the addition of the Aβ peptide that accumulates during Alzheimer's disease (AD) (Stein et al., 2004). Injection of inhibitory antibodies to TTR into a transgenic animal model for AD results in the loss of neuroprotective functions of TTR and acceleration of the disease process (Stein et al., 2004). Thus, the loss of TTR function hastened disease pathogenesis in an animal model of a neurodegenerative disease. Finally, genetic variants of TTR cause transthyretin-associated hereditary amyloidosis (ATTR) in which amyloid accumulates in various tissues and organs (Bergen et al, 2004). ATTR is most common between the third and seventh decades of life, similar to that of ALS. During ALS, it is likely that reduced CSF levels of TTR result in altered transport of T4 and retinol/vitamin A, thus altering thyroid function and increasing oxidative stress. It is also probable that reduced TTR levels in motor neurons decrease neuroprotective functions and increase oxidative stress in motor neurons, thus enhancing neurodegeneration. In addition, TTR genetic variants may increase susceptibility of individuals to develop ALS or hasten disease pathogenesis.
Transthyretin (TTR) variants can be identified by mass spectrometry and verified by gene sequencing. For the mass spectrometry, TTR first is purified from a human sample by affinity chromatography using anti-TTR antibody. The purified TTR is reduced using tris(2-caroxyethyl)phosphine (TCEP) and then analyzed by mass spectrometry to resolve differences in the protein mass indicative of a polymorphism in the TTR gene resulting in an altered amino acid. The most common variants (denoted as wildtype amino acid, location within the protein, and mutant amino acid) are Val30Met and Gly6Ser, though over 100 variants are known (see, e.g., Connors, 2003). Specific mass shifts observed by mass spectrometry are indicative of gene polymorphisms. Samples containing such variant proteins can be analyzed by DNA sequencing to confirm the mass spectrometry results. TTR genetic variants may increase susceptibility to developing ALS due to environmental exposures.
Recent studies have shown that transthyretin functions as a neuroprotective protein and injection of transthyretin protein reduces amyloid deposition and cognitive decline in an animal model of Alzheimer's disease (Stein, 2004). Transthyretin protein levels are reduced by exposure to heavy metals and other toxins (Zheng, Toxicol. Sci., 2001; Zheng, Microscopy Res. & Tech., 2001). Environmental exposure to lead and other heavy metals has been proposed as a risk factor in the etiology of ALS (Vinceti, 1997; Kamel, 2002). Genetic variants of transthyretin decrease protein function and cause a familial form of polyneuropathy (Benson, 1985). ALS subjects that have transthyretin genetic variants that decrease transthyretin function will make the individual at increased risk for developing ALS, thus suggesting that transthyretin is a novel risk or susceptibility factor for ALS. It is likely that the transthyretin plays a role in motor neuron survival and genetic polymorphisms and/or decreased expression levels make neurons more susceptible to injury or toxin exposure. This could help explain the increased incidence of ALS in Gulf War Veterans deployed to specific active zones noted above. TTR protein may also directly participate in motor neuron degeneration by generating extracellular or intracellular toxic protein aggregates. TTR fibrils have been observed in familial polyneuropathies induced by TTR mutations (Sousa, 2003). In vitro studies have shown that the toxic form of TTR is the non-fibrillar protein aggregates and that fully formed TTR containing fibrils is non-toxic (Sousa, 2001; Sousa, 2002). Therefore the presence of non-fibrillar TTR protein aggregates in ALS spinal cord tissue will be interpreted as directly contributing to motor neuron degeneration. These experiments provide novel data implicating TTR and TTR functional pathways in motor neuron survival and highlight new pathogenic mechanisms for ALS. Reduced TTR function, either by genetic polymorphisms or post-translational modifications that alter protein function or by reduced expression levels during ALS, may directly induce motor neuron cell loss since the loss of retinoid signaling can induce motor neuron disease in rodents (Corcoran, 2002).
The second neuroendocrine protein is 7B2 (Martens, 1989). It has been determined that 7B2 protein alterations occur during ALS. More specifically, in ALS subjects increased levels of a carboxy-terminal fragment of 7B2 referred to as 7B2CT were observed. 7B2 is a neuroendocrine secretory protein that functions as a chaperone for the proprotein convertase 2 protein (PC2). 7B2 binds to PC2 in the endoplasmic reticulum and facilitates its transport to other compartments of the secretory pathway where it is proteolytically matured and activated (Mbiday, 2001). PC2 participates in the production and secretion of numerous hormones and neuropeptides, including neuropeptide Y, somatostatin, galanin, and vasopressin. A prior study has shown expression of 7B2 in motor neurons and in the spinal cord. 7B2 also acts as a chaperone protein for the maturation and secretion of insulin-like growth factor 1 (IGF-1), which is currently in trials as a therapy for ALS (Chaudhuri, 1995). 7B2 is processed by the enzyme furin to form a carboxy-terminal fragment called 7B2CT that functions as an inhibitor of PC2 activation. Carboxypeptidase E cleaves 7B2CT for its degradation. Furin and/or carboxypeptidase E activity may also be altered in ALS subjects. Therefore increased levels of 7B2CT in the CSF of ALS subjects may indicate that PC2 activation is reduced in multiple cell types of the nervous system, including motor neurons. This implies that maturation and secretion of many neuroendocrine peptides, growth factors and hormones is reduced in ALS.
The third neuroendocrine protein identified is cystatin C (Abrahamson, 1990). Cystatin C has been identified using mass spectrometry as a diagnostic biomarker for ALS. CSF and lumbar spinal cord tissue samples from ALS subjects exhibit less cystatin C than control subjects. Cystatin C is a secreted protein that functions both as a cysteine protease inhibitor and can function as an autocrine or paracrine factor in neurogenesis of neural stem cells. Mutations in the cystatin C gene cause a rare disease called hereditary brain amyloid angiopathy, and increased levels of cystatin C have been found in other neurodegenerative diseases including Alzheimer's disease, ischemia, and Creutzfeldt-Jakob disease (CJD). Decreased levels of cystatin C in the CSF of ALS subjects or altered post-translational modifications to cystatin C suggest decreased levels of protease inhibitors, which may contribute to disease pathogenesis.
Despite the identification of early protein biomarkers for ALS, there remains a need, however, for improved methods for identifying therapeutic targets of ALS, and improved methods of diagnosing and monitoring the progress of the disease.